Abstract: Drugs targeting the spindle assembly checkpoint (SAC), such as inhibitors of Aurora kinase B (AURKB) and dual specific protein kinase TTK, are in different stages of clinical development. However, cell response to SAC abrogation is poorly understood and there are no markers for patient selection. Methods A panel of 53 tumor cell lines of different origins was used. The effects of drugs were analyzed by MTT and flow cytometry. Copy number status was determined by FISH and Q-PCR; mRNA expression by nCounter and RT-Q-PCR and protein expression by Western blotting. CRISPR-Cas9 technology was used for gene knock-out (KO) and a doxycycline-inducible pTRIPZ vector for ectopic expression. Finally, in vivo experiments were performed by implanting cultured cells or fragments of tumors into immunodeficient mice. Results Tumor cells and patient-derived xenografts (PDXs) sensitive to AURKB and TTK inhibitors consistently showed high expression levels of BH3-interacting domain death agonist (BID), while cell lines and PDXs with low BID were uniformly resistant. Gene silencing rendered BID-overexpressing cells insensitive to SAC abrogation while ectopic BID expression in BID-low cells significantly increased sensitivity. SAC abrogation induced activation of CASP-2, leading to cleavage of CASP-3 and extensive cell death only in presence of high levels of BID. Finally, a prevalence study revealed high BID mRNA in 6% of human solid tumors. Conclusions The fate of tumor cells after SAC abrogation is driven by an AURKB/ CASP-2 signaling mechanism, regulated by BID levels. Our results pave the way to clinically explore SAC-targeting drugs in tumors with high BID expression.

Abstract: The RAS-regulated RAF–MEK1/2–ERK1/2 (RAS/MAPK) signaling pathway is a major driver in oncogenesis and is frequently dysregulated in human cancers, primarily by mutations in BRAF or RAS genes. The clinical benefit of inhibitors of this pathway as single agents has only been realized in BRAF-mutant melanoma, with limited effect of single-agent pathway inhibitors in KRAS-mutant tumors. Combined inhibition of multiple nodes within this pathway, such as MEK1/2 and ERK1/2, may be necessary to effectively suppress pathway signaling in KRAS-mutant tumors and achieve meaningful clinical benefit. Here, we report the discovery and characterization of AZD0364, a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and kinase selectivity. In vitro, AZD0364 treatment resulted in inhibition of proximal and distal biomarkers and reduced proliferation in sensitive BRAF-mutant and KRAS-mutant cell lines. In multiple in vivo xenograft models, AZD0364 showed dose- and time-dependent modulation of ERK1/2-dependent signaling biomarkers resulting in tumor regression in sensitive BRAF- and KRAS-mutant xenografts. We demonstrate that AZD0364 in combination with the MEK1/2 inhibitor, selumetinib (AZD6244 and ARRY142886), enhances efficacy in KRAS-mutant preclinical models that are moderately sensitive or resistant to MEK1/2 inhibition. This combination results in deeper and more durable suppression of the RAS/MAPK signaling pathway that is not achievable with single-agent treatment. The AZD0364 and selumetinib combination also results in significant tumor regressions in multiple KRAS-mutant xenograft models. The combination of ERK1/2 and MEK1/2 inhibition thereby represents a viable clinical approach to target KRAS-mutant tumors.

Abstract: Background: Aurora kinases (AurK) represent potential targets for anticancer therapy in hematological malignancies and solid tumors. AurK B inhibitor AZD1152 (barasertib) showed benefit (35% CR/CRi) in patients (pts) with untreated AML when given as a 7-day continuous infusion (Lowenberg B et al, Blood 2011, Kantarjian HG et al., Cancer 2013). AZD2811NP, a nanoparticle encapsulated slow-release inhibitor of AurKB, when given as 2-4hr IV on days 1 and 4, offers a prolonged drug exposure in vivo, mimicking the AZD1152 7-day continuous IV infusion. This is an update on the first-in-man dose-escalation study of AZD2811NP in pts with relapsed/refractory AML/MDS or treatment-naïve patients (pts) not eligible for intensive induction therapy (NCT03217838). The primary objectives are to determine the Maximum Tolerated Dose (MTD) and safety profile of AZD2811NP monotherapy and in combination with azacitidine. The secondary objectives are to evaluate the pharmacokinetic (PK) profile, Biologically Effective Dose (BED), and preliminary efficacy (CR, CRi, PR, 6 month OS). Methods: Pts received a 2-hour IV infusion on Day 1 and 4 of each 28-day cycle (Cy) for doses up to 600mg, extending to a 4 h IV infusion for dosages 〉 600 mg. In the ongoing dose escalation, 3-6 pts have been sequentially enrolled in cohorts ranging from 100 mg to 800 mg per infusion (Day 1 & 4), i.e. from 200 mg to 1,600 mg per cycle in monotherapy setting, according to a modified continuous reassessment method (mCRM) dose escalation design. AZD2811NP was also combined with azacitidine (75 mg day 1 to 7 or the 5-2-2 schedule) starting at an AZD2811NP dose of 400 mg D1 and D4 every 4 weeks. Study treatment was continued until disease progression, intolerability, or when discontinuation criteria were met. Results: Currently, 30 pts have enrolled of which 29 pts (12 females and 17 males) received study treatment in 5 monotherapy cohorts and 2 azacitidine combination cohorts, with ages ranging from 53 to 85 years. Nineteen pts had relapsed/refractory AML, 9 pts had MDS and 1pt had MDS/MPN. Monotherapy cohort 5 (800 mg D1 & D4) and combination cohort 4c (600mg D1 & D4 + Azacitidine) are currently enrolling. Of the 19 pts in monotherapy cohorts 1-5, 18 pts discontinued (due to consent withdrawal [2], early disease related deaths [2] , other reason [1], or completed follow up [13; 11 pts after Cy1, 2 pts after Cy2] ) and 1 pt is still on therapy. Nine pts were treated in combination with azacitidine, and of these, 3 pts are still on therapy and 6 pts have discontinued AZD2811NP (due to death [1], consent withdrawal [2] , or completed follow up [3; 2 pts after Cy2, 1 pt after Cy4]). Adverse events that occurred in ≥ 20% of pts were mainly myelotoxicity, nausea and fatigue. One dose-limiting toxicity (DLT) has been observed in the monotherapy arm (esophageal infection) and one DLT in the combination setting (late neutropenia recovery). Two deaths were due to the underlying disease and 1 due to a serious adverse event of Gr 5 sepsis not related to study drug. AZD2811 total and released blood PK exposure appears broadly dose proportional with a terminal t1/2 of ~ 30-50 hours. Released blood PK exposure is ~ 1% of total PK exposure. Conclusion: AZD2811NP is documented to be well tolerated at doses up to 600 mg on Day 1 & 4 every 28 days in monotherapy setting and up to 400 mg (D1 & 4) in combination with azacitidine. The monotherapy and combination therapy dose escalations are ongoing. Updated results including preliminary efficacy data will be presented. Additional dose finding and expansion cohorts of AZD2811NP in combination with venetoclax are planned. Disclosures Atallah: Pfizer: Consultancy; Helsinn: Consultancy; Jazz: Consultancy; Helsinn: Consultancy; Novartis: Consultancy; Takeda: Consultancy, Research Funding; Jazz: Consultancy. Yang:AstraZeneca: Research Funding; Agios: Consultancy. Eghtedar:Jazz: Consultancy, Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; Verastem Oncology: Consultancy; Novartis: Consultancy, Honoraria, Speakers Bureau. Borthakur:Merck: Research Funding; Oncoceutics: Research Funding; Cantargia AB: Research Funding; FTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Argenx: Membership on an entity's Board of Directors or advisory committees; Xbiotech USA: Research Funding; Arvinas: Research Funding; Polaris: Research Funding; Strategia Therapeutics: Research Funding; Tetralogic Pharmaceuticals: Research Funding; Agensys: Research Funding; Bayer Healthcare AG: Research Funding; AstraZeneca: Research Funding; BMS: Research Funding; Eli Lilly and Co.: Research Funding; PTC Therapeutics: Consultancy; NKarta: Consultancy; BioLine Rx: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cyclacel: Research Funding; GSK: Research Funding; Janssen: Research Funding; Incyte: Research Funding; AbbVie: Research Funding; Eisai: Research Funding; Novartis: Research Funding; BioTheryX: Membership on an entity's Board of Directors or advisory committees; Oncoceutics, Inc.: Research Funding. Charlton:AstraZeneca: Employment; GSK: Equity Ownership. MacDonald:AstraZeneca: Employment, Equity Ownership. Korzeniowska:AstraZeneca: Employment. Sainsbury:AstraZeneca: Employment, Equity Ownership. Strickland:Sarah Cannon Development Innovations: Employment. Overend:AstraZeneca: Employment, Equity Ownership. Adelman:AstraZeneca: Employment, Equity Ownership. Fabbri:AstraZeneca: Employment. Travers:AstraZeneca: Employment. Smith:AstraZeneca: Employment, Equity Ownership. Pease:AstraZeneca: Employment, Equity Ownership. Cosaert:AstraZeneca: Employment. OffLabel Disclosure: AZD2811NP, a nanoparticle encapsulated slow-release inhibitor of Aurora Kinase B (AurKB), is an investigational agent in clinical trials for human cancers including AML/MDS.

Abstract: Background: Aurora kinases represent potential targets for anticancer therapy in solid tumors and hematological malignancies. In a phase I/II study, the aurora B kinase inhibitor AZD1152 (barasertib) showed benefit (35% CR/CRi) in patients (pts) with untreated AML when given as a 7-day continuous infusion (Lowenberg B et al, Blood 2011, Kantarjian HG et al., Cancer 2013). AZD2811 nanoparticle is a novel, encapsulated slow-release inhibitor of Aurora kinase B offering several advantages compared with AZD1152, including prolonged drug release in vivo. AZD2811 nanoparticle mimics the AZD1152 7-day continuous infusion when given as a 2-hr infusion on Day 1 and 4, and resulted in increased efficacy and decreased toxicity in vivo. We report the first-in-man dose-escalation of AZD2811 nanoparticle in pts with relapsed AML/MDS or treatment-naïve patients not eligible for intensive induction therapy. The objectives were to determine the safety profile, MTD, PK, dosing schedule and preliminary efficacy of AZD2811 nanoparticle. Methods: Patients received a single 2-hour IV infusion on Day 1 and 4 of each 28-day cycle. Based on the previous experience with AZD1152 in the same patient population, the expected MTD is in a range of 1,200 mg per cycle. In the ongoing dose escalation, cohorts of 3-6 patients have been sequentially enrolled in 4 cohorts ranging from 100 mg to 600 mg per infusion on Day 1 and 4 every 28 days, i.e. from 200 mg to 1,200 mg per cycle. Patients were allowed to continue to receive AZD2811 until disease progression, tolerability, or discontinuation criteria have been met. The study uses a Bayesian adaptive design approach to dose escalation to improve the efficiency and precision of the MTD estimation compared to a traditional 3+3 design. Results: Currently, 10 pts with age ranges from 56 to 86 years have completed DLT assessment period for the first 3 cohorts. 9 patients had relapsed/refractory secondary AML/MDS after failure of hypomethylating agents and 1 patient had a relapsed, therapy-related AML. Cohort 4 (600 mg per infusion D1 and D4) is currently under evaluation. Of the 10 pts in cohorts 1-3, 7 pts discontinued due to disease progression, 1 discontinued due to subject decision/consent withdrawal, 1 discontinued due to physician decision, and 1 pt is active and ongoing. AEs assessed as related to AZD2811 that occurred in one or more patients were Grade 3/4 neutropenia/thrombocytopenia, Grade 3 anemia and Grade 1 fatigue, rash and stomatitis. Thus far, no DLTs and no fatal AEs related to AZD2811 have been observed. 2 deaths have been reported, 1 due to the underlying disease and 1 due to a Serious Adverse Event of Gr 5 Sepsis (not related to study drug). AZD2811 total blood PK appears dose proportional with a t1/2 of 30-50 hours. Conclusion: AZD2811 nanoparticle is safe and well tolerated at a dose up to 400 mg on Day 1 and 4 every 28-days. The monotherapy dose escalation is ongoing and updated results including preliminary efficacy data and supporting preclinical data will be presented. Additional dose finding and expansion cohorts of AZD2811 nanoparticle in combination with azacytidine and venetoclax are planned. Disclosures Atallah: Novartis: Consultancy; Abbvie: Consultancy; BMS: Consultancy; Jazz: Consultancy; Pfizer: Consultancy. Charlton:AstraZeneca: Employment. MacDonald:AstraZeneca: Employment. Young:AstraZeneca: Employment. Sainsbury:AstraZeneca: Employment. Overend:AstraZeneca: Employment. Adelman:AstraZeneca: Employment. Travers:AstraZeneca: Employment. Smith:AstraZeneca: Employment. Pease:AstraZeneca: Employment. Brugger:AstraZeneca: Employment.

Abstract: Pim kinases have been shown to play a key role as downstream effectors of growth factor signaling in hematological malignancies. We have previously described AZD1208, a novel, orally bioavailable, highly selective pan Pim kinase inhibitor, undergoing clinical testing in AML. Specifically, we had demonstrated the anti-proliferative activity of AZD1208 in models of AML and had shown that AZD1208 treatment of AML cell lines results in the dose dependent reduction of pBAD at serine 112 and p4EBP1 at serine 65 as well as pp70S6K at threonine 389 and pS6 at serine 235/236, associated with global effects on protein translation (Keeton et al. Blood 2014). Since AZD1208 is about 10-15 fold more potent against Pim-1 than Pim-2 in both enzymatic and cellular assays, we sought to assess the dependency of each of these markers on Pim-1 and/or Pim-2 for phosphorylation using a Pim-1/3 selective inhibitor (Pim-1/3) that lacks Pim-2 inhibitory activity. In vitro studies demonstrate that while the modulation of pBAD does not require inhibition of Pim-2, the regulation of 4EBP1 as well as p70S6K and S6 are dependent, at least in part, on Pim-2 activity. As these proteins are canonically regulated by mTORC1, these data are consistent with Pim-2 acting upstream of this complex; perhaps through phosphorylation of TSC2 as reported for multiple myeloma cell lines (Lu et al. Blood 2013). These observations are also supported by in vivo data. Analysis of pBAD and p4EBP1 levels in Molm16 xenografts show that maximal inhibition of pBAD (70-80% inhibition) is achieved at AZD1208 plasma concentration of about 1000 ng/ml, consistent with the estimated cellular IC90 of pBAD inhibition in vitro. However, maximal inhibition of p4EBP1 is achieved at concentrations 3-5 fold higher. This observation is consistent with the differential potency of AZD1208 against Pim-1 and Pim-2, and with a requirement for Pim-2 inhibition for maximal p4EBP1 inhibition in this AML model. pBAD and p4EBP1 were selected as the pharmacodynamic endpoints for evaluating clinical target inhibition in patient samples. Bone marrow and peripheral blood samples were collected pre- and post-dose from relapsed, refractory AML patients enrolled in the AZD1208 Phase I dose escalation. Analysis of pBAD inhibition on day 1 of dosing shows at least 60-70% inhibition across all of the dosing cohorts. With clinical exposures on day 1 approaching 1000 ng/ml at the first dose level and exceeding 1000 ng/ml as the dose increases, the extent of pBAD inhibition seen in patients appears to be consistent with Pim-1 inhibition, as seen in preclinical models. Furthermore, and also similar to our preclinical observations, maximal inhibition of p4EBP1 in patients is achieved only at higher exposures. These data strengthen the hypothesis that BAD phosphorylation is primarily dependent on Pim-1, whereas suppression of Pim-2 activity is required for maximal inhibition of p4EBP1 in AML cells. In summary, Pim inhibition in AML cell line models and in patients treated with AZD1208 results in the inhibition of the downstream targets of Pim signaling, pBAD and p4EBP1. Invitro and in vivo, the inhibition of pBAD is consistent with inhibition of Pim-1 while inhibition of p4EBP1 indicates a requirement for Pim-2 inhibition as well. These observations are validated in patients and provide further evidence for the relevance of these biomarkers as a measure of Pim signaling in AML. Disclosures McEachern: AstraZeneca: Employment, Equity Ownership. O'Connor:AstraZeneca: Employment, Equity Ownership. DuPont:AstraZeneca: Employment, Equity Ownership. Gibbons:AstraZeneca: Employment, Equity Ownership. Pablo:AstraZeneca: Employment, Equity Ownership. Vishwanathan:AstraZeneca: Employment, Equity Ownership. McCoon:AstraZeneca: Employment, Equity Ownership. Cortes:AstraZeneca: Research Funding. Neumann:AstraZeneca: Employment, Equity Ownership. Keating:AstraZeneca: Employment, Equity Ownership. Pease:AstraZeneca: Employment, Equity Ownership. Brown:AstraZeneca Pharmaceuticals: Employment, Patents & Royalties. Barrett:AstraZeneca: Employment, Equity Ownership.

Abstract: Therapeutic resistance to frontline therapy develops rapidly in small cell lung cancer (SCLC). Treatment options are also limited by the lack of targetable driver mutations. Therefore, there is an unmet need for developing better therapeutic strategies and biomarkers of response. Aurora kinase B (AURKB) inhibition exploits an inherent genomic vulnerability in SCLC and is a promising therapeutic approach. Here, we identify biomarkers of response and develop rational combinations with AURKB inhibition to improve treatment efficacy. Experimental Design: Selective AURKB inhibitor AZD2811 was profiled in a large panel of SCLC cell lines (n = 57) and patient-derived xenograft (PDX) models. Proteomic and transcriptomic profiles were analyzed to identify candidate biomarkers of response and resistance. Effects on polyploidy, DNA damage, and apoptosis were measured by flow cytometry and Western blotting. Rational drug combinations were validated in SCLC cell lines and PDX models. Results: AZD2811 showed potent growth inhibitory activity in a subset of SCLC, often characterized by, but not limited to, high cMYC expression. Importantly, high BCL2 expression predicted resistance to AURKB inhibitor response in SCLC, independent of cMYC status. AZD2811-induced DNA damage and apoptosis were suppressed by high BCL2 levels, while combining AZD2811 with a BCL2 inhibitor significantly sensitized resistant models. In vivo, sustained tumor growth reduction and regression was achieved even with intermittent dosing of AZD2811 and venetoclax, an FDA-approved BCL2 inhibitor. Conclusions: BCL2 inhibition overcomes intrinsic resistance and enhances sensitivity to AURKB inhibition in SCLC preclinical models.

Abstract: The RAS/MAPK pathway is a major driver in oncogenesis and is dysregulated in approximately 30% of human cancers, primarily by mutations in BRAF or RAS genes. The extracellular-signal-regulated kinases (ERK1 and ERK2) serve as key central nodes within this pathway. The feasibility of targeting the RAS/MAPK pathway has been demonstrated by the initial clinical responses observed to BRAF and MEK inhibitors in BRAF V600E/K metastatic melanoma, however resistance frequently develops by reactivation of the pathway. Direct targeting of ERK1/2, may provide another therapeutic option in tumours with mutations in BRAF or RAS genes. Importantly, ERK1/2 inhibition may have clinical utility in overcoming acquired resistance to RAF and MEK inhibitors where RAS/MAPK pathway reactivation has occurred, such as relapsed BRAF V600E/K melanoma. Starting from our published work,1 we will describe for the first time, a scaffold hopping approach leading to the identification of AZD0364, a pre-clinical ERK1/2 inhibitor candidate drug. Driven by conformational modelling and structure-based design, and by utilising novel sulfamidate ring opening chemistry, a high lipophilicity efficiency core was identified. Structure based, multi-parameter based optimisation of this improved core ultimately led to AZD0364. AZD0364 exhibits high cellular potency against a direct downstream substrate on the MAPK pathway (e.g. inhibition of phospho-p90RSK1 in BRAFV600E mutant A375 cells, IC50 = 6 nM). The molecule is a highly selective kinase inhibitor (10/329 kinases tested are inhibited at & gt;50% at a 1 µM) and has long residence time on the protein (as determined by SPR on human unphosphorylated-ERK2: pKd = 10; t1/2 = 277 mins). The good in vitro potency and selectivity is complemented by excellent physico-chemical properties (maximum absorbable dose estimated to be & gt;4 g) and good oral pharmacokinetics across species, leading to a low predicted dose to man. In xenograft models, AZD0364 inhibits phospho-p90RSK1 in tumors in a dose-dependent manner. AZD0364 induces regressions in the KRAS mutant NSCLC Calu 6 xenograft model. AZD0364 can also be combined safely and effectively with the MEK1/2 inhibitor selumetinib in KRAS mutant NSCLC xenograft models. 1Richard A. Ward et. al. Structure-Guided Discovery of Potent and Selective Inhibitors of ERK1/2 from a Modestly Active and Promiscuous Chemical Start Point, J. Med. Chem. 2017, 60, 3438−3450. Citation Format: Iain Simpson, Mark J. Anderton, David M. Andrews, Jason Breed, Emma Davies, Judit E. Debreczeni, Vikki Flemington, Francis D. Gibbons, Mark A. Graham, Philip Hopcroft, Tina Howard, Julian Hudson, Clifford D. Jones, Christopher Jones, Nicola Lindsay, J Elizabeth Pease, Philip Rawlins, Karen Roberts, Steve Swallow, Steve St-Gallay, Michael E. Tonge, Richard A. Ward. Discovery of AZD0364, a potent and selective oral inhibitor of ERK1/2 that is efficacious in both monotherapy and combination therapy in models of NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1647.

Abstract: Neuropeptide Y (NPY) is thought to play a key role in stimulating feeding, thus making NPY receptors attractive appetite suppressant drug targets for treating obesity. Because the orexigenic effects of NPY have been ascribed to actions at the NPY Y5 receptor, we have determined the role of this receptor in feeding in rats, using a small molecule antagonist of this receptor. NPY5RA-972 is a selective and potent ( & lt;10 nmol/l) NPY Y5 receptor antagonist. This compound is central nervous system (CNS) penetrant, and an oral dose of 10 mg/kg NPY5RA-972 to rats produced concentrations in cerebrospinal fluid that Xgreatly exceeded the in vitro IC50 (inhibitory concentration 50%). Indeed, at doses to rats as low as 1 mg/kg, NPY5RA-972 inhibited feeding induced by intracerebroventricular (ICV) administration of a selective NPY Y5 agonist ([cPP1–7,NPY19–23,Ala31,Aib32,Gln34]-hPP). However, in the dose range 1–10 mg/kg, NPY5RA-972 had no significant effect on food intake in Wistar rats induced to feed by either ICV NPY or 24 h fasting or in free-feeding Wistar or obese Zucker rats. Chronic administration of NPY5RA-972 (10 mg/kg twice daily) had no effect on food intake or body weight in either free-feeding Wistar rats or dietary obese rats. These data indicate that NPY5RA-972 is a potent, selective, orally active, and CNS-penetrant antagonist of the NPY Y5 receptor that prevents feeding driven by activation of this receptor. The data obtained with this antagonist indicate that the NPY Y5 receptor is not a major regulator of feeding in the rat.